Trastuzumab (Herceptin), a humanized anti-HER2 monoclonal antibody, has been demonstrated to confer a clear survival benefit when it is added to standard chemotherapy for HER2-overexpressing breast cancer. Unfortunately, however, only 30-40% of HER2-positive breast tumors respond to trastuzumab. In this proposal, we will explore whether erythropoietin (EPO)-induced cell signaling plays a role in breast cancer resistance to trastuzumab. EPO is a prime hematopoietic cytokine produced mainly by a group of specialized cells in the kidney and acts on erythroid progenitor cells in the bone marrow, leading to erythropoiesis. Recombinant human EPO (rHuEPO) is frequently used in cancer patients to prevent and alleviate cancer- and cancer treatment-related anemia and fatigue. Recent studies have shown that EPO has remarkable cytoprotective activity in nonhematopoietic tissues via activation of the EPO receptor (EpoR) found in these tissues. Interestingly, EpoR is also present in some clinical tumor specimens, particularly tumor specimens from breast cancer patients. As a member of the type I cytokine receptor family, EpoR homodimerizes upon EPO binding, triggering activation of the receptor-associated kinase Jak2, subsequent phosphorylation of STAT5, and a signal transduction cascade mediated by SH2 domain-containing adaptors that overlaps substantially with HER2-mediated signaling pathways. We found in our preliminary studies that EpoR is co-expressed with HER2 in a significant number of established breast cancer cell lines and breast cancer tissue specimens from patients. Exposure of breast cancer cells to rHuEPO activated cell signaling in an EpoR expression-dependent manner, and concurrent treatment of HER2/EpoR dual-positive breast cancer cells with trastuzumab and rHuEPO reduced the therapeutic response to trastuzumab treatment. We hypothesize that cell signaling in response to concurrent administration of rHuEPO constitutes a clinically relevant mechanism of breast cancer resistance to trastuzumab. We plan to test this hypothesis in four specific aims: 1) to confirm the cytoprotective effect of rHuEPO against trastuzumab in HER2/EpoR dual-positive breast cancer cells; 2) to determine the extent to which concurrent administration of rHuEPO counteracts the antitumor activity of trastuzumab in HER2/EpoR dual-positive breast cancer xenografts; 3) to elucidate the EpoR downstream signaling pathways through activation of which rHuEPO mediates cytoprotection against trastuzumab; and 4) to confirm the presence of HER2 and EpoR co-expression and to determine its frequency in tumor specimens from breast cancer patients. Attainment of these aims will reveal whether EPO/EpoR signaling contributes to trastuzumab resistance. A positive finding will warrant further investigation of whether rHuEPO should continue to be administered concurrently with trastuzumab in breast cancer patients. PUBLIC HEALTH RELEVANCE: The drug erythropoietin (EPO) is often given to breast cancer patients to help prevent or lessen treatment-related anemia and fatigue; however, some evidence from our laboratory and others suggests that EPO might make breast cancer resistant to the anti-breast cancer drug trastuzumab. We will investigate this possibility. Our findings may indicate that EPO should not be administered to breast cancer patients who are being treated with trastuzumab.